CA1110467A - Methods to determine a diagnostic indicator of blood sugar conditions and liquid chromotographic columns therefor (cyanide free) - Google Patents
Methods to determine a diagnostic indicator of blood sugar conditions and liquid chromotographic columns therefor (cyanide free)Info
- Publication number
- CA1110467A CA1110467A CA363,487A CA363487A CA1110467A CA 1110467 A CA1110467 A CA 1110467A CA 363487 A CA363487 A CA 363487A CA 1110467 A CA1110467 A CA 1110467A
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- column bed
- particles
- suspension
- equilibrated
- test sample
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
- H01M50/00—Constructional details or processes of manufacture of the non-active parts of electrochemical cells other than fuel cells, e.g. hybrid cells
- H01M50/30—Arrangements for facilitating escape of gases
- H01M50/342—Non-re-sealable arrangements
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
- H01M50/00—Constructional details or processes of manufacture of the non-active parts of electrochemical cells other than fuel cells, e.g. hybrid cells
- H01M50/10—Primary casings, jackets or wrappings of a single cell or a single battery
- H01M50/172—Arrangements of electric connectors penetrating the casing
- H01M50/174—Arrangements of electric connectors penetrating the casing adapted for the shape of the cells
- H01M50/182—Arrangements of electric connectors penetrating the casing adapted for the shape of the cells for cells with a collector centrally disposed in the active mass, e.g. Leclanché cells
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
- H01M50/00—Constructional details or processes of manufacture of the non-active parts of electrochemical cells other than fuel cells, e.g. hybrid cells
- H01M50/30—Arrangements for facilitating escape of gases
- H01M50/375—Vent means sensitive to or responsive to temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
- G01N30/6004—Construction of the column end pieces
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
- Y02E60/10—Energy storage using batteries
Abstract
ABSTRACT
Methods and microcolumns to determine a numerical percentage value as a diagnostic indi-cator of the blood sugar condition of a specific person. A whole blood sample is lysed and a test sample thereof is placed on a microchromatographic column bed of ion exchange material particles. The column bed is characterized by having no cyanide therein. The column bed comprises an equilibrated suspension of particles selected from the class con-sisting of CarXH and CarYOH, where "Car" repre-sents an inert substrate for carrying ionizable groups X- providing dissociated cations H+ and ionizable groups Y+ providing anions OH-. Fractions of various hemoglobin species in the test sample are preferentially eluted or desorbed from the column bed by buffer and wash solutions character-ized by having no cyanide therein. Amounts of hemo-globin species in the eluate fractions are detec-ted and measured by spectrometric (color) analy-sis. A mathematical computation using integer factors corresponding to amounts of various hemo-globin species produces the numerical percentage value.
Methods and microcolumns to determine a numerical percentage value as a diagnostic indi-cator of the blood sugar condition of a specific person. A whole blood sample is lysed and a test sample thereof is placed on a microchromatographic column bed of ion exchange material particles. The column bed is characterized by having no cyanide therein. The column bed comprises an equilibrated suspension of particles selected from the class con-sisting of CarXH and CarYOH, where "Car" repre-sents an inert substrate for carrying ionizable groups X- providing dissociated cations H+ and ionizable groups Y+ providing anions OH-. Fractions of various hemoglobin species in the test sample are preferentially eluted or desorbed from the column bed by buffer and wash solutions character-ized by having no cyanide therein. Amounts of hemo-globin species in the eluate fractions are detec-ted and measured by spectrometric (color) analy-sis. A mathematical computation using integer factors corresponding to amounts of various hemo-globin species produces the numerical percentage value.
Description
METHODS TO DE'I`ERMINE A DIA&NOSTIC INDICATOR
(~f;` BL.OOD SUGAR CONDITIONS, AND ~ LIQUIl~
CHROMATOGRAPHIC COLUMNS T~IEREI~OR (CYAI~IDE FREE) TECHNICAL FIEI.D
The inven~ion relates to methods -to de-termine a numerical percentage value as a diagnos-tic indicator of the blood sugar condition of a specific person. The invention also relates to im--proved liquid chromatographic columns having no cyanide therein for practice of the methods.
BACKGROUND ~.Rl~
__ _ __ ~ccorclLng t:o t~le lnvclll.ion, a whoLe b].oo(l ~ample 1~ collected Fr~m Itle person or pcl~:lcrlt rlrlcl thereilEter pr.eptlrecl a.9 a re(l bLoo(l ceLl helllolysLlte test sample us:Lng alternative clinical chemistry techniques and procedures. Thereafter, the inven-tlon provides a series of steps for separating, detecting, and measuring the amount of a group oE
hemoglobin species present in the test sample us-ing improved ion exchange liquid column microchroma-tographic apparatus, techniques and procedures"
spectrometric analysis, and mathematical comp~lta-t:ions, .tn biochem:lstry, hemog,l.obi.ns are the am~
photerlc protein molecule coloring matter of t'ne red blood corpuscles serving to convey oxygen to the tissues. Several chromatographically separable minor hemoglobins are presen-t in red blood cell hemolysa~es of normal persons. Some minor hemo-globins are designated as Hb-Ala Hb-Alb Hb-ALC
Hb-Ald and Hb-Ale The hemoglobin species Hb-AlC
.
.. . . .
" ' '. ' , ' ` :
~' ~................................. ' ' ' . . .
: -is most prominent and accounts for the ~ajor por-tion of the minor hemoglobins. It is l;nown t~at the level of hemoglobin ~b-AlC has been related ~o a patient's average blood sugar level. Normal per-sons are expected to have 3-6C/o Hb-AlC relative to their total hemoglobin. Untreated diabetics may have 6 12% Hb-AIC relative to their total hemoglo-bin, whether the affliction is of the juvenile-onset or adult-onset type. Sti].l further, it is understood that the levels oE the species Hb-AlC, as a separate and identifiable sub-group, may serve as an indicator of the degree of hypergly-cemia, an excess of sugar in the blood, over a pro-longed period of time.
The assignee of the present invention, Isol~b, Incor~orated, i8 now the owner oE Eive United S~ates patents rel~ting to metho(ls ~nd colu~ns similar to the methocls and columns disclosed herein. These patents are: No. ~ 2,85'j; No. 4, 142,856; No. 4,142,857; and ~o. 4,142,858, each granted ~Iarch/1979 to Acuff; and No. 4,168,147, September/1979, also to Acuff.
The first three referenced Isolab-Acuff prior art patents ('~55, '~56 and '857) use an ion exchange column comprising an equilibrated suspen-sion of cellulose particLes. The cellulose parti-cles are of a weak base and anion exchange type in '855; oE a weak acid and cation exchange type in '856 and '857. The last ~wo reEerencecl Isolab-Acuff prior art patents ('858 and '147) use an ion exchange column of an equilibrated suspension of resin particles. The resin particles are of a weak acid and cation exchange type. .
In each of the Isolab-Acuff patents, reEerence is made to a "~treatmen~ solution" 28 ' .
.
,.
... . . . . . ..
.
. ,-" .' .' , .. ' '' , ~ ' ',' . : .. . ~ ..
, . .: . .: , ' . .
~; ,, : . ;: :
.. . .
used to prepare the ion exchange particles 27 in the ~orm of an equilibratecl suspension 27~S). In each pacent, the treatment solution 28, st-litaL~îy adjusted for pH and/or lonic strerlgth, is also used as the "elution solutionl' 28 or 128, added ~o the column 20 after introduction of -the tes~ sample 10. Each prior treatment solu-tion 28 and el~tion solution 28 or 128 has been charac~eriæed by t~e presence of a cyanide compound or anion ~N .
The use of cyanide (KCN) as an ingr~d-ient or active compound in buffers or developers for hemoglobin chromatograms has long been accepted as essential; cle riguer. In the ~rio-r l.;.~er~lture, a p~per by Allen et al., Obxervcltions on ~lle Cllrorn.l-tograph:i.c Heterogeneity o~ Norm.lL Adu:lt an(l l:le~
~lum~n ~lemoglobi.n ~ S~ucly oE the EfE~ s a~ld C't:'y~
st.ll.lization and ~hromato~.r,rclplly On the ~le~erogenc~i-ty ancl IsoLeuci~le Content, Jowrnal of the _erican Chemical Society, VoL. 80, pp. 1628-1634, April 1958, discloses that "~p]otassium cyanide was ori-ginally included in the developers in order ~o de-crease the dissociation oE Eerrihe~oglobin cyanide during chromatography. It was -not removed ~rom the developers when oxyhemo~lobin was chrorn,~l-o-graphed because ~errihemogLobin cyanide ancl oxy-he~o~lobin have :ident:ical chromcltographic behavior.
Thus, traces o~ ~err:LheMoglob:ln (methelTIogLohin) in sol~tions of oxyhemoglobin are converted to ferrihemoglobin cyanide and do not produce slow moving extraneous zones on the column." supra. p.
1630.
Subsequent ~o the inventions described in the Isolab-Acuff patents, it has been cliscovered that microchromatographic cl:inical techrliques and procedures to determine an indication or level of :
. , . ~ "
the hemovlobin species ~Ib-Ala_c do not require presence oF a cyani.de compound or radical., in either a particle treatment solution or a buE-Eer or elution solution. Indeed, thle presence oE
cyanide may be considered as del~eterious.
By way of illustration, each of the Iso-lab-Acuff patents discloses t'nat the amount oE hemo-globin species in a particular eluate fraction, particularly Hb-Ala c is detected and meas~red by spectrometric (color) analysis. Each patent spe-cifically discloses that the spectrometric analysis may be performed by an apparatus 40 which measures absorption of light caused by the hemoglobin spe-cies present in the test sample 10. The pr.ior patents rely upon the known fact that the vis:ible portion oE the spectrum Eor detect:i.ng tlle presence oE a hemoglo~in is in the biolet rangel more speci.-fically, at substan~:ially 415 nm or ~1.50 A.
After filing of the applications pre-ceding the Isolab-Acuff patents, it became apparen-t that the integers expressing the amounts of hemo-g~obin species present i.n each eluate Er~ction (as detennined by spectrometric analysis at 415 nm) were affected by a factor of time. ~tlhen the spec-trometric analysis was performed rather promptLy (e.g., 30 mlnutes af~.er el-.ltion), the resu.Ltclrlt :Lnteger woulcl con.~orm to a norm or starld~l-rcl. Ilow-ever, after a :I.onger perio(l of tilllC (e.&., 60 minutes), the resultant integer would reflect a lower value for the hemoglobin species Hb-Al~ c After careEul analysis of all aspects of the methods and columns disclosed in the Isolab-Acuff patents, it has now been determined, and is there-fore specified as being critical, that the presence of a cyanide compound or radical contribu-ted to an inaccuracy in the true or reasonably correct in-teger reflecting the hemoglobln species ~Ib-Al~ r present in a particular and specific tes~ sample.
It is no~ understood that the presence of cyanide in either the i.on exchange partlcle treatment sol~tion or the buffer solution will in-crease the incidence of a tirr.e-factored conversion of ferrous hemoglobin deri.va~ives to errihemo~
globin cyanide, with an attendant alteration in the integer or value obtained by spectromet-ric analysis.
Of course, a skilled laboratory ~erson o-r techni-cian would (and wiLl) compensate for such varia-tion. However, ~rovision o~ a standard test -that can (and wîll) be ~erfor~ed by relatively unskille(l personnel mandates the u.se of a techn:ique ancl pro-cedure which does not em210y cyan:ide.
Other advant..l~e~s o~ rlon-(lse o~ cyanide w:il.l incl.ude the Eacil:LtatLon of export-:irrlport:
of mi.crocol~l~ms and rea~,ents accorcling ta the :inven-tion. In many countries throu~hout the world~
labeling of a ~roduct as containin~ cyanide (even in trace amounts) presents reg1l1atory complications.
In countries of Droduct manu:Eacture, eliminati.on oE
wastes or residue re:Lating to either reagen~ pre-paration or ion exchange material equilibra~ic)n can (and will) give cause to environmental llaYclrd situat:ions and legit:imate gc~vernmental entity con-cern. Finally, disposal of used colurnns and reagent solutions is simpLif:ied.
DISCLOSURE OF INVENTION
It is an object of the invention to ~ro-vide methods to determine a nu~erical percentage value as a diagnostic indicator of the blood sugar , ~ .
. : . ,; :
.
condition of a speclfic person, witho~lt the use o.E
reagents or solutinns containing any amounts, trace or otherwise, of any cyanide compound ~r anion C~ .
It is a further object to provide a S method which will separate, detect and measure the ratio of the sub-group of hemog]obitl species Hb-A-la c to the total hemoglobins (Hb) present in the blood of a specific person quickly, inexpensively, accur-ately and without the presence of a cyanide com-pound or the anion CN .
It is still further an object to provide a method which, a].though reyuiring oE a number of sequential or consecutive steps, is of such a charac-ter and nature as to permit the adopt:ion o.E pr()-cedures arlcl pro~ocoLs ~Ih3.ch may becorne st.ln~ (l .. nn(l ro~ltlne, p~rm:itt:in~ per.son~ sk:i.lled in the a-r~ o.f.
c1inical. chem:lstry to rcpeat:ecll.y an~l cncc~lr~:lt:eLy test the bloocl o.E large groups of persons to estab-lish a data base for use by qualified, specialized and medically trained personnel in diagnosing the blood sugar condition of specific persons suspected as being diabetic.
It is still another object of the inven-tion to provide microcolumns, for chromatographic clinical techniques and procedures to determ:ine an lndlcation or level. of the hemo~rjl.o~:in species ~Ib-~la c having no cyanlcle therein but: h.lv:ing prede~-termineclrnicrochrornatograph:Lc char.lcter:istics and properties for practice of the rnethods according to the invention.
These and other objects of the invention, as well as the advantages thereof, will be apparent in view of the detailed descriptions of the various embodiments thereof as set :Eorth below~
In general, the methods according to ~he invention to determine a numerical percentage value as a diagnostic indicator of the blood s~gar condi-tion of a specific person use a whole blood sample taken from such person and -thereafter prepared as a test sample containing a red b3ood cell hemoly-sate solution. Thereafter, a quanti~y of the test sample is introduced into an end of a column bed having no cyanide therein which will absorb hemo-globin species present in sa;d test sample. The column bed comprises an equilibrated s~lspension of ion exchange material particles havlng a size less than 100 mesh, The particles in a particular column bed are one member selected from the class consisting of CarX~ arl(l CarYOIl, "Car" represents an inert substrat:e Eor carryi.~ .i.oni.~,al)Lcl gro~ll)s X
l.S providing c~ oc~ ed ca!:.i.ons ll arld iotl:iz~)l.)Le ~,,roup~ Y~ provi~ling di~ns.soci.at~(l r.lniot-~c; 01~ .
I~ ha9 been detertll~ined as cri~i.cr.ll.and essential for the practice of the invention that the CarXH particles be weakly acidic cation ex-changers and have a reported pKa: 3-7 and be used in an equilibrated suspension at a pH: 6.0-75 al:
22.5C.
It has also been determi.ned ~s criticrl:l and esse~ 'L ror ~ l~c~ l-r<lc~: i.cc ol~ tl~ velll:. i.OI~
tha~ the CarYO~l parti.cles be wealil.y hLI~;i.c ~Ini.on exch~mger.s cltlcl llave .~ report.ed pK,I: 7-1.0 r.lnd be usecl in an equi'l.ibrated suspens.ion at a p~l: 7.'3-9.0 at 22.5C, Therea:Eter a quantity of a buEfer solu-tion having no cyanide therein is introduced into an end of the column bed to preferentially elute therefrom a first fraction which contains certain of the hemoglobin species present in t~e test sample. t~hen the column bed is an ec~ui.Li.brc.Lted suspension of CarX~I particles, the ~irst :Frc.lct-ion " ~.$~
will contain substantially all of the hemoglobin species Hb-Ala c present in the test sample. ~hen the column bed is an equilibratecl suspension of CarYOH particles tne first fraction will contain essentially all of the ~hemoglobin species present in the test sample other than, to the exclusion of, Hb-A]a c In either event~ an al.iquo~ quantity o~
a first eluate fraction is collected :Erom the other end of -the column bed.
In the ~irst method embodiment of the in-vention, a quantity of a wash solution is lntro-duced into an end of the column bed ~o desorb and elute therefrom a second fraction ~hich will con-tain substantially all o:E the remaining hemoglobin lS species present in the test sampl.e. ~hen ttle column bed :is an equilibrated suspeTIsion oE CarXtl pc~rt:icles, the c;ecorl(l I:'ract:ion w:i. I.L c.~ont:cl:i.rl es~qcr~ y a L ~
~h~ hemo~:Lob:lrl species preserlt ln the tes t sanlp.l.e~
other than, to the excluslon o.E, Hb-Ala c ~Jhen the column bed is an equilibrated suspension of CarYOH particles, the second fraction will contain s~bstantially all of the hemoglobin species Hb-Ala c present in the test samp:le In ei~her event, an aliquot quantity of ~ second e:Luate fraction is collected from the other end of the column bed.
Then, tlle hemoglobin speci.es present in the .ELrst and second eluate fractions are separately detected and measurecl ~y s~ectrc~metric an~nlysi.s (color) and the resrJective ar.~ounts ~hereof are ex-pressed as numerical values ~h.ich are then compared in accordance with a mathematical Eorrnula.
In the second method embodiment of the invention, using a column bed having an equilibra-ted suspension of either CarXH or CarYOII particles and a buffer solution, an aliquot quanti~y of a first eluate frac~ion is collected from an end of the column bed. Also, a quantity of a test sample is significantly diluted to provide a red blood cell hemolysate solution ~hich m,~y be conveniently detected and measured by spectrome-tric analysis.
Then, the hemoglobin species present in the first eluate fraction and in the diluted hemolysate sol-u-tion are separately detected and meas~red by the spectrometric analysis (color) and the respective amounts thereoE are expressed as numerical values which are then compared in accordance with a rnathe-matical formula.
In e:ither method e~bodiment, iln appro-priate mathematical ~ormuLa wlll provi(le a nullleri-L5 cal percentclge vaLuc Eor thc hemogLobln sp~cies ~Ib-Ala c present in ~ particuLclr te,C;t sampLe Eor use as a diagnostic indicator of the blood sugar characteristics of the person providing that test sample.
In all embodiments of the inVentiOTl, the resultant numerical percentage value is available for use by qualified, specia:Lized and ~edically trained personnel as a diagnostic indicator of the blood sugar characterist:ics oE tile patien~ p-rovicl-ing thc test sample.
A microcolumn Eor use in the pract:ice of either embodiment o~ ~he inventiorl has a reservoir discharging into a barrel terminating in a dis-charge tip. The junctures between the reservoir and barrel and the barrel and discharge tip are closed by ~ransverse discs. Ion exchange parti-cles positioned in the barrel between ~he discs provide a column bed. The discs are permeable to a red blood cell hemolysate solution. The column bed comprises an equilibrated sus~ension of parti-.
.
cles having no cyanide therein. The particles will have a size of less than 100 mesh and in a particu-lar column will have one member selected from the class consisting of CarX~. and CarYOH.
BRIEF_DESCRIPTION OF ~RAI~TII~GS
The drawing schematically shows practice of the invention s~ecifically as to the first method embodiment wherein first and second fractions of hemoglobin species are eluted from an improved chromatographic microcolumn shown substantlally in full scale.
BEST MO ~ FOR C R YING OUT 'l'llt~'. tNV~l'lON
Practice oL~ tLIe me~hocl .:lccord:i.ng ~.o ~he lnventl.orl reclulres tlle coL:Lec~i.on o~ ~1 whol.e bloocl sample from a person or patienr.. The whole blood sample may be taken using conventionaL clinical chemistry techniques and procedures.
The disclosure o~ the Isolab-Acuf~ prior art patents describing in detail two general sl:a~e of the art procedures for preparing a suitab~Le test sample, referred to generally by the numeral 10 containing a red blood cell hemolysate solution Erom a whol.e bloocl samp:le, I.s :incorporatecl herc:iTl by reEerence. A proceclurc LO-a is used to prepclre a test sample LO which is predominantly the hemo-globin content of the whole blood sample. A pro-cedure 10-b is used to prepare a test sample 10 which may include ~he plasma proteins, lipids~ and -the white and red blood cell debris, in addition to the hemo~lobin content of the whole blood sarnple.
~lith reference to the drawing, which is substantia].ly in full scale, a chroma-tographic microcolumn is indicated general'Ly by the numeral 20. A column 20 comprises a reservoir 21 discharg-ing into a barrel 22 ter~inating in a discharge tip 23 selectively closed by a cap 24 The juncture or intersection between the rese]rvoir 21 and the barrel 22 is closed by a transverse plate or disc 25. The juncture between the barrel 22 and the dis-charge tip 23 is also closed by a transverse plate or disc 2~. The ion exchange materiaL particles comprising the column bed between the discs 25 and 26 are referred to generally by the numeral 27.
Each retaining disc 25 and 26 is permea-hle, h.lving a networl; o~ mi.c-ropores perm~ irlg introduc~lon oE a red b'lood ce'Ll hemoly~;ate so':Lll-tion ~rom res~rvo:ir ~l into the barreL 22, arld re-moval of an clu.lte :Eract:ion :~rom the barrc!.l. 22 through the tip 23, while retaining the colulTm bed of particles 27 within the barrel 2~. The discs 25 and 26 may be made from a conventional flexible, resilient, linear, high density polyethylene o~ the Ziegler type. Com~ercially, th:is type oE fllter grade polyethylene is produced and sold under the name of Vyon.
According to the inVentiQn, t'he ion ex-change materl.al parti.cles 2'7 have a si.æe less than 'l00 mesh and are selectcd ~rom the class consisting of Car~H and CarYO~I. "Car" represents an inert substrate and may be cellulosic or a resin copoly-mer oE polystyrene or rnethacrylic acid and divinyl-benzene. X represents an ionizable group provid-ing dissociated cations H~ carried by a "Car" and may be a carboxyl group or a carboxymethyl group.
Y represents an ionizable group providing disso-ciated anions OH carried by a "Car" and may be a .
~. .. .
diethylaminoethyl group or a mix~ure of amine groups having the general for~ulae - NH,, N~.R ancl M(R)2.
The CarXH particles 27 may also be charac-terized as weakly acidic cation exchangers having a reported pKa: 3-7. The CarYOH particles 27 may also be charac~erized as weakly basic anion ex-changers having a reported pKa:7-lO.
The commercially available form of ion exchange material particles 27 will usually require preparation or ~rea~.ment for use ln a barrel 22 of a microcolumn 20 between the discs 25 and 26. Such treatment could be perEormed with the particl.es 27 in situ in the column barrel 22. Ilowever, i.t i.s pre~errecl thnt the particL,es 27 ~.'or ~I, scri.e(; of `L5 .i.clenticaL columnc; 20 be t.reate(l us:ing a ~at:ch ~chnl.~lu~, wh:L~Il wi.Ll pe~rlrlLL t:he usc o~ .).la.lmrll: 2() havi.n~ preclel:ermine(l mi.crochromatog,r~ll)h:ic ctl~lrac-teristics ancl properties.
The ion exchange material particles 27 constituting the column bed of a column 20 are used in the form of an equilibrated suspension 27(S) h,aving a predetermined or "starting" pH. The CarXH
particles are used in a suspension 27(S~ at a pH:
6.0-7.5 at 22.5C. The CarYOll ~art-icl.c~s .Ire ~Ised in a suspension 27(S) a~. a p~-l: 7.'3-9.0 at 22.5C.
A suspenslon 27(S) :Ls prepared us:ing a treat:ment solution 2~, having no cyanide therein.
According ~o the invention, a treatment solution 28 may also be used as buffer solution 128 for elution from a column bed, comprising a sus-pension 27(S) of either CarXH or CarYOH particles, oE an eluate fraction containing certain, but not all, of the hemoglobin species present in the test sample 10.
A quan~i-ty of a test sample 10 prepared by either procedure 10-a or 10-b is introduced into one end of a column 20 having a coltlmn bed com-prising an equilibrated suspension 27(S). A test sample prepared according to procedure 10-a will require a 1:4 dilution using dislilled water.
Preferably, the column 20 is positioned vertically, the discharge tip cap 24 is removed and a predetermined volume of a test sample 10 is dis-charged or placed into the reservoir 21. A mclJor portion of the test sample 10 will pass readil.y through the disc 25 and onto t~le column bed ol the suspension 27(S). The minor portion of the test sample 10 remaining on or i.n the disc 25 slloul.d be purged or clisplaced onto the co:L~Irnn bed usinF~ a small volume (e.g., 0.2 ml) of a soLu~:i.on :in~ (k!cl :~or u~e ac; a buEfer ~oLut::ion 128 in tlle n~-~xt se~luell-tial metllocl ~;teE) accorclinf~ to t~e :invent:iorl.
A predetermine~ or aliquot quantity o.E a buffer solution 123 having no cyanide therein is dis-charged into the column reservoir 21 to preferential-ly elute from the discharge tip 23 a first .Eraction 30 of the test sample lØ The fi:rst fraction 30 i5 collected in a receiver 31. I~hen the column bed i.s a suspension 27(S) of CarX}I particles, the fi.rst fraction 30 ~ill contain substantially all of the hemoglobin specie~ llb-~La c present i.n ~.he test sample 10. When the colunm bed :is a suspension ~7(S) o Car~OH partic:les, the first fraction 30 wi:Ll con-tain essentially all of the hemoglobin species pre-sent in the test sample 10 other than, to the ex-clusion of, Hb-Ala c In either event, after a period of time following addition of the buffer 28, an aliquot quantity of a first fraction 30 will be collected in a receiver 31.
In the firs-t method embodiment of the in , : ~
.,, ' ' ~, $ 7 vention, as shown in the drawing, a second fraction 32 of the test sample 10 is collected. in a receiver 33 after an aliquot quanti~y of a wash solution 34 is discharged into the col-umn reservoir 21. The S second fraction 32 will contain subs~antially all of the remaining hemoglobin species present in the test sample 10. ~hen the column bed is a suspension 27(S) of CarXH par~icles, the second fraction 32 will contain essenti.ally all of the hemoglobin spe-cies present in the test sample 1.0 other than llb-Ala c ~Jhen the column becl is a suspension 27~S) of CarYOH par~icles, the second fraction 32 will con-tain substantially all of the hemoglobin species Hb-Al~ c present in the test sampl.e 10. In either event, an alicluo~ uant~ y o~ a. secorlcl Eract.ion 32 will be co~ cte~ i.n a r~ceI.ver 3~.
~r~,~. wash so:l.ut:.i.on ~4 w:ill. hllve llO ey~l~li.(le~
th~reln. Otherwise, ~ precise .~orn~ula oE a wasll solution 34 is not critical, so long as use thereof will not alter or modify the spectrometric absorp-tion characteristics ("color") of an elute fractlon 32. A compatible wash sol.ution 34 will have either ionic strength or relative pH suEficient to f~ Ly or completely desorb substantiaLly all the rem11n-ing blood components of a test sample 10 Erom a column bed of ion exchange material particles 27.
For example, 1 four milliliter (4 ml) vol~lme of l~M
NaCl m&y be discharged into a colurnn reservoir 21.
After a period of time (e.g., 20-30 minutes), an eluate fraction of substantiaLly 4 ml volume will be collected in a receiver 33. A fraction 32 eluted from a suspension 27(S) of CarXH particles will require of suitable dilution using distillecl water prior to the next sequential methocl step according to the invention.
According to both method embodiments of the invention, the determination of tlle ra-~io of Hb-Ala c to the to~al hemoglobins (Hb) present in the whole blood sample collec-ted from the person or patient uses spectrometric apparaLtus referred to generally by the numeral 40, following performance of liquid column microchromato~raphic techniques and procedures using a test sample 10.
The spectrometric analysis is perEormed by an apparatus 40 which measures absorption of li~ht caused by the hemoglobin species present -in the test sample lO. It is known ~hat the visible portion of the spectrllm for cletectin~J the preC;ence 0 f rl tle mo ~ l ob :irl L s :in ~ e vi.ole~ r an ~.~,c.~ 0 ~
LS speci~:lcal.l.y, at s~ st~n~::Lally ~5 tlm ol ~L5() ~.
Th~ aL~paratus ~t) may be an c)pt i..cr~ JI)ec~-trometer "~cclic-lte(l" or pre-set at the seLectecl wave length of 4l5 nm. The apparatus 40 may also be a spectrophotometer, a form of spectrometer with associated equipment which supplies the ratio, or a function of the ratio, of the radiant power of two beams as a function of an acljustably selec-ted spectral wave length. Because the spectrome-tric analysis accorcling to the invention :is ~or the purpose of detecting ancl measuli.n~ llen~o~.~:lobi.n species Erorn the test .sampLe 1.0 by li.~ht absorE~-tion characteristics, altcrnative Forms of appara-tus 40 could b~ used; for example, visual compara-tors such as a set of Nessler tubes.
In the first method embodiment of ~he in-vention, the contents of the receivers 31 and 33 are individually transferred into appropriate cu-vettes for the spectrometric apparatus 40. Spec-trometric analysis of the first and second eluate fractions 30 and 32 will provide integers or , natural nwmbers which will express, represent or indicate the amounts of hemoglob-in species present in the test sample 10. When using a conventional spectrometer or spectrophotomete:r as the apparatus 40, the displayed interger is a function of the absorbance ~A), a measurement of the amount of light of the spectral wave leng-th of 415 nm absorbed by the hemoglobin species during passage through the cuvette and toward the sensing photocell.
The expressed numerical -values for the separately detected and measured hemoglobin species in each eluate fraction 30 and 32 oE a par-ticular test sample 10 are then compared in accordance with a mathematiccll formula.
When the eLuate ;~ractic)ns 30 arld 32 are eluted ~rom a colu~m hecl comprisln~, a ~nlsl)en-sLon 27(S) o~ CarXH par~lcles, thc comp~a~ioLl Ls inte~er for firs-t fraction 30 x 100 =
integer for first fraction~30 -~
integer for second fraction 32 a numerical percentage value.
When the eluate Eractions 30 and 32 are eluted from a column bed comprising a swspen-sion 27(S) of CarYO~I particles, the c(mlputclt:io nt~ or ~:~cond_Lraction 32 x 100 integer i-or ~rirsl~ fraction 3() -~
integer Eor second fraction 32 a numerical percentage value.
In the second embodiment of the inven-tion, using a column bed comprising a suspension 27(S) of either CarXH or CarYOH particles and a buffer solution 128, analiquot quan~ity of a first eluate fraction 30 is collected in a receiver 31.
Also, either at a prior time, concurrently or con--17~
secutively, a quantity of the test sample 10 is prepared as a red blood cell hemolysate solution 42 which may be conveniently det,ected and measured by spectrometric analysis. It will be apparent that the light absorption characteristics of -the test sample 10, without significant dilution~ would be of such magnitude as to impair operation e~ficiency of the sensing photocell of conventional spectro-metric apparatus 40. Accordingly, and by way of example, a quantity of test sample 10 equal to the volume of test sample 10 introduced into an end of a column 20, prior to elution oE the first ~raction 30, showld be cliluted using distilled water in the ratio substantially l:l~8o to prepare a hemolysate lS ~solution 42 Eor analysl~ by a ~:pcctrome~ric aE~para-tus l~O.
The exl)resse.d n~lmerica'l. Vcll.Ue~ Eor the separately cletecte~ atld measured hernoglobLn spec:ies in the eluate fraction 30 and in the diluted herno-lysate solution 42 oE a particular test sample lO
are then compared in accordance with a mathemati-cal formula.
When the eluate fraction 30 is eluted from a column bed comprising a suspells:Lon 27 (S) of CarXH particles, the computation is integer for eluate ~raction. 30 x 100 in te ger for flelTIo'r--ate ~sol~
a n~erical percentage value.
When the eluate fraction 30 is eluted from a column bed comprising 27(S) of CarYOH par-ticles, the computation is (inte~er for eluate fraction 30 x 100) _ ~ ~ (integer for hemolys'ate'sorution ~
a numerical percentage value.
Use any of these mathematical compu--tations will provide a nwmerical value :Eor the hemoglobin species Hb-Ala c present in a particu-lar test sample 10 for use as a diagnostic indica-tor of the blood sugar character:istics of -,he per-son providing that test sample.
The -following Examples will ~urther illustrate and describe the practice of t~e inven-tion by persons skilled in the art of clinica]
chemistry.
Exarnple 1 A CarYOH ion exchange material particle 27 may be of the type wherein "Car"is cellulosic and Y~ Ls a die~hylaminoethy:L group, -O-Cll2-C~l2-N(C~l5~2, having a reported pK,I oE substanti~ll.Ly 9.5. Commerckllly, ~his lype oE iOLI (~xch~ e m~
terial ls sol(l under the ~rlme Whatman DEAE-52.
These cellulose particles 27 are pre-pared as a suspension 27ts) having a pH of sub-stantially 8.5 at 22.5C by mixing with a "tris"
treatment solution 28 of -the fo~lula 6.06g H2N.C
(CH2OH)3 (0.05 M), with 0.10g NaN3 (0.01%) as a preservative, made up in one liter of H20. After mixing with the treatment solution 28, the cel:lu-lose particles 27 are then Eurther treated w:ith an acid solution 29 (e.g., 4 M HCl) to readjust to the pH of substantially 8.5.
In both method ernbodiments of the in-vention, a quantity of a buffer solution 128 is introduced into an end of a column bed of a sus-pension 27(S)of these cellulose particles to pre-f~rentially elute therefrom an eluate fraction 30 containing essentially of the hemoglobin species present in a test sample 10 other than Hb-Ala_C.
The buffer solution 128 may be a "tris" solution of the same formula as the -treatment solution 28 wlth the pH adjusted to substantially 7.7 at 22.5C
using concentrated HCl.
' ,, ~
Another CarYOH ion exchange material particle 27 may be of the type w'herein "Car" ls a resin copolymer of polystyrene and divinylbenzene and Y+ is a mixture of primary, secondary, and ter-tiary amine groups having the general :Eormulae -NH2, N~'~ and N(R)2, where R is an aliphatic hydro-carbon radical such as -CH3 or -C2H5. These par-: ticles 27 have a reported pKa: 7-9. Cor~mercially, this type of ion exchange material is sold under the names Dowdex M~A-l and Amberlite IRA-93.
These resi.n part:ic'les 27 may be pre-pared as a suspension 27(S) llaving a pl~ OL s~lb-stanti~'lly 7.5 a~ ~.2..5C ~y m:l.xitlg wi.~ c~ ri.~
trcatment ~o'lu~Lon 28 o.E the ~ame .f.'orm-lLcl flS UXCIII1-ple 1.
A first fraction 30 of a .est sample 10, containing essentially all of the hemoglobin species other than Hb-Al,l c' may be elu~ed from a column bed of a suspension 27(S) o~ these resin particles by use of a "~ri.s" bufEer so.lLl~:ioll 1.28 of the same formula as in ~xample 'l, w.i~h a pH ad-justed to substantially 7.5 at 22.5C.
Exame~e 3 A CarXH ;.on exchange material parti-cle 27 may be of the type wherein "Car" i5 a resin copolymer of methacrylic acid and divinylbenzene and X+ is a carboxyl group, -COOH, having a reported pKa: ~-6. Commercially, this type of ion ex-change material is sold under the name Amberlite , :. ' " ' ' ~ ' ' :, ', ' : . , . .. - : .
:.: ' : ' .. , ' ' .' . ', ,, . ', ., :
The resin polymers 27 are prepared as a suspension 27(S) having a pH of substantialy 6.98 at 22.5C by mixing wi~h "phosphate" treatment solution 28 of the formula: 3.74g KH2PO4 (0.027M) 7 0.955g KOH (0.017 M), with 0.10g NaN3 (0.01%) as a preservative, made up in one liter of H20. After mixing with the treatment solution 28, the resin particles 727 are then further treated with an acid solution 29 (e.g., 4 M H3PO4) to readjust to the pH of substantially 6.98.
In both method embodiments of the inven-tion, a quantity of a bu~fer solution 128 is intro-duced into an end of a column bed of a suspenslon 27(S) of these resin particles to preferentially elute therefrom an eluate fractlon 30 containing substantially aLl of the hemoglobi-n species Hb-Ala c present ln a test sample 10. The buffer so lutic)n 128 may be a "phosphate" solutioll o~ th~
same orm-l1a as the treatment solutlon 28 antl hav~
ing a pH of substantially 6.98.
Examp~e 4 Another CarXH ion exchange material particle 27 may be of the type wherein "Car" is cellulosic and X is a carboxymethyl group, -O-CH2-COOH, having a reported PKa of 3.5. Commer-clally, this type of ion exchange mater:ial :i.s solcl under the nam~ Whatman CM-52.
These cellulose particles 27 may be prepared as a suspension 27(S) having a pH of sub-stantially 6.8 a~ 22.5C by mixing with "phosphate"
treatment solution 28 o~ the formula: 3.74g KH2PO4 (0.027 M), and 0.748g KO~ (0.013 M), with 0.10g NaN3 (0.01%) as a preservative, made up ln one liter of H20.
,. . - . :
, $ ~
A first frac-~ion 30 of a test sample 10, containing substantially all of ~he hemoglobin species Hb-Ala_C, may be eluted from a colu~n bed of a suspension 27(S) of these cellulose particles by use of a "phosphate" buffer solution 128 of the same formula as -the treatment solution 28 and having a pH of substantially ~.8.
_Example 5 The cellulose particles 27 o:E Example 4 may be prepared as a suspension 27(S) having a pH of substantially 6.1 at 22.5C by mixing with a "bis-tris" treatment solution 28 of the formula:
6-28g ~HOCH2c~l2)2 NC (Ctl2O~1)3 ~0.03M), with O.lOg lS NaN3 (0.01%) ~s a preservatlve, mad~ up i.n one liter o~ ~l20. ~Eter mixLng, wi~h the treat:merlL- ~o:lalti(3n 28, the cellulose particles 27 are ~hen fuL~:her treated with an acid solution 29 (e.g., 4 M HC]) to readjust to the pH of substantially 6.1.
A first fraction 30 of a test sample 10, containing .substantially all of the hemoglobin species ~Ib-AIa c~ may be eluted from a column bed of a suspension 27(S) of these cellulose part:icles by the use of a "bis-tris" buffer solution l28 of ~5 the same formuLa as the treatment solutlon 28 with the addltion of 2.34g NaCl (0.04 M) ~ncl having a p~l of substantially 6.1.
Example 6 The resin particles of Example 3 may be prepared as a suspenslon 27(S) having a pH of substantially 6.8 at 22.5C by mixing with a "bis-tris" treatment solution 28, of the same formula as in Example 5. A first fraction 30 of a tes-~
sample 10, containing substan-tially all of the hemoglobin species Hb-Ala c~ may be eluted from . . ..
, ' ~,'; ,' ' . ' ' '"
': ' .. ,, ' ' a column bed of a suspension 27('~) of these resin particles by the use of a "bis-trisll buffer solu-tion 128 of the same formula as t:he treatment solu-tion 28 with the addi~ion of 7.02g NaCl (0.12M), having a pH of substantially 6.8.
SU~ARY
In all embodiments of the inven-tion as described, several steps, techniques or proce-dures are disclosed wherein dilution, usin~ dis-tilled water, is either required or suggestecl. It will be undexstood by a person skilled in the art of clinical chemistry tha~ the best modes of prac-ticing the invention usin~, an i.rnproved IllicrocoLurnn 20 will recluire careElll a(loptl()rl an~l corlsis~:c?nL
followlng of rout:ine proce(lures, i.E ~lle inVerl~
i~s tt) xepre8ent a rel kLbl~e metho(l of assess.in~
the presence oE d:Labetes and monitoring the degree of diabetic control. It will be further understood by a practitioner of the invention that a procedure or protocol for repetitive testing of large numbers oE persons, both diabetic and normal, will inherent~
ly incorporate therein: standard quantities and volumes of test samples 10, solutions 28 and ]28, 29 cmd 34, Erac~ions 30 and 32 and sol-l~ion 42;
conslstent and compatible dilut:ion ratios; and, care~ul select:ion and re~ lation of the sl~ectro-metric apparatus 40. There~ore, the Eull scope and extent of the invention should be determined solely by the words of the claims appended hereto.
.. . ~ .
' ' '
(~f;` BL.OOD SUGAR CONDITIONS, AND ~ LIQUIl~
CHROMATOGRAPHIC COLUMNS T~IEREI~OR (CYAI~IDE FREE) TECHNICAL FIEI.D
The inven~ion relates to methods -to de-termine a numerical percentage value as a diagnos-tic indicator of the blood sugar condition of a specific person. The invention also relates to im--proved liquid chromatographic columns having no cyanide therein for practice of the methods.
BACKGROUND ~.Rl~
__ _ __ ~ccorclLng t:o t~le lnvclll.ion, a whoLe b].oo(l ~ample 1~ collected Fr~m Itle person or pcl~:lcrlt rlrlcl thereilEter pr.eptlrecl a.9 a re(l bLoo(l ceLl helllolysLlte test sample us:Lng alternative clinical chemistry techniques and procedures. Thereafter, the inven-tlon provides a series of steps for separating, detecting, and measuring the amount of a group oE
hemoglobin species present in the test sample us-ing improved ion exchange liquid column microchroma-tographic apparatus, techniques and procedures"
spectrometric analysis, and mathematical comp~lta-t:ions, .tn biochem:lstry, hemog,l.obi.ns are the am~
photerlc protein molecule coloring matter of t'ne red blood corpuscles serving to convey oxygen to the tissues. Several chromatographically separable minor hemoglobins are presen-t in red blood cell hemolysa~es of normal persons. Some minor hemo-globins are designated as Hb-Ala Hb-Alb Hb-ALC
Hb-Ald and Hb-Ale The hemoglobin species Hb-AlC
.
.. . . .
" ' '. ' , ' ` :
~' ~................................. ' ' ' . . .
: -is most prominent and accounts for the ~ajor por-tion of the minor hemoglobins. It is l;nown t~at the level of hemoglobin ~b-AlC has been related ~o a patient's average blood sugar level. Normal per-sons are expected to have 3-6C/o Hb-AlC relative to their total hemoglobin. Untreated diabetics may have 6 12% Hb-AIC relative to their total hemoglo-bin, whether the affliction is of the juvenile-onset or adult-onset type. Sti].l further, it is understood that the levels oE the species Hb-AlC, as a separate and identifiable sub-group, may serve as an indicator of the degree of hypergly-cemia, an excess of sugar in the blood, over a pro-longed period of time.
The assignee of the present invention, Isol~b, Incor~orated, i8 now the owner oE Eive United S~ates patents rel~ting to metho(ls ~nd colu~ns similar to the methocls and columns disclosed herein. These patents are: No. ~ 2,85'j; No. 4, 142,856; No. 4,142,857; and ~o. 4,142,858, each granted ~Iarch/1979 to Acuff; and No. 4,168,147, September/1979, also to Acuff.
The first three referenced Isolab-Acuff prior art patents ('~55, '~56 and '857) use an ion exchange column comprising an equilibrated suspen-sion of cellulose particLes. The cellulose parti-cles are of a weak base and anion exchange type in '855; oE a weak acid and cation exchange type in '856 and '857. The last ~wo reEerencecl Isolab-Acuff prior art patents ('858 and '147) use an ion exchange column of an equilibrated suspension of resin particles. The resin particles are of a weak acid and cation exchange type. .
In each of the Isolab-Acuff patents, reEerence is made to a "~treatmen~ solution" 28 ' .
.
,.
... . . . . . ..
.
. ,-" .' .' , .. ' '' , ~ ' ',' . : .. . ~ ..
, . .: . .: , ' . .
~; ,, : . ;: :
.. . .
used to prepare the ion exchange particles 27 in the ~orm of an equilibratecl suspension 27~S). In each pacent, the treatment solution 28, st-litaL~îy adjusted for pH and/or lonic strerlgth, is also used as the "elution solutionl' 28 or 128, added ~o the column 20 after introduction of -the tes~ sample 10. Each prior treatment solu-tion 28 and el~tion solution 28 or 128 has been charac~eriæed by t~e presence of a cyanide compound or anion ~N .
The use of cyanide (KCN) as an ingr~d-ient or active compound in buffers or developers for hemoglobin chromatograms has long been accepted as essential; cle riguer. In the ~rio-r l.;.~er~lture, a p~per by Allen et al., Obxervcltions on ~lle Cllrorn.l-tograph:i.c Heterogeneity o~ Norm.lL Adu:lt an(l l:le~
~lum~n ~lemoglobi.n ~ S~ucly oE the EfE~ s a~ld C't:'y~
st.ll.lization and ~hromato~.r,rclplly On the ~le~erogenc~i-ty ancl IsoLeuci~le Content, Jowrnal of the _erican Chemical Society, VoL. 80, pp. 1628-1634, April 1958, discloses that "~p]otassium cyanide was ori-ginally included in the developers in order ~o de-crease the dissociation oE Eerrihe~oglobin cyanide during chromatography. It was -not removed ~rom the developers when oxyhemo~lobin was chrorn,~l-o-graphed because ~errihemogLobin cyanide ancl oxy-he~o~lobin have :ident:ical chromcltographic behavior.
Thus, traces o~ ~err:LheMoglob:ln (methelTIogLohin) in sol~tions of oxyhemoglobin are converted to ferrihemoglobin cyanide and do not produce slow moving extraneous zones on the column." supra. p.
1630.
Subsequent ~o the inventions described in the Isolab-Acuff patents, it has been cliscovered that microchromatographic cl:inical techrliques and procedures to determine an indication or level of :
. , . ~ "
the hemovlobin species ~Ib-Ala_c do not require presence oF a cyani.de compound or radical., in either a particle treatment solution or a buE-Eer or elution solution. Indeed, thle presence oE
cyanide may be considered as del~eterious.
By way of illustration, each of the Iso-lab-Acuff patents discloses t'nat the amount oE hemo-globin species in a particular eluate fraction, particularly Hb-Ala c is detected and meas~red by spectrometric (color) analysis. Each patent spe-cifically discloses that the spectrometric analysis may be performed by an apparatus 40 which measures absorption of light caused by the hemoglobin spe-cies present in the test sample 10. The pr.ior patents rely upon the known fact that the vis:ible portion oE the spectrum Eor detect:i.ng tlle presence oE a hemoglo~in is in the biolet rangel more speci.-fically, at substan~:ially 415 nm or ~1.50 A.
After filing of the applications pre-ceding the Isolab-Acuff patents, it became apparen-t that the integers expressing the amounts of hemo-g~obin species present i.n each eluate Er~ction (as detennined by spectrometric analysis at 415 nm) were affected by a factor of time. ~tlhen the spec-trometric analysis was performed rather promptLy (e.g., 30 mlnutes af~.er el-.ltion), the resu.Ltclrlt :Lnteger woulcl con.~orm to a norm or starld~l-rcl. Ilow-ever, after a :I.onger perio(l of tilllC (e.&., 60 minutes), the resultant integer would reflect a lower value for the hemoglobin species Hb-Al~ c After careEul analysis of all aspects of the methods and columns disclosed in the Isolab-Acuff patents, it has now been determined, and is there-fore specified as being critical, that the presence of a cyanide compound or radical contribu-ted to an inaccuracy in the true or reasonably correct in-teger reflecting the hemoglobln species ~Ib-Al~ r present in a particular and specific tes~ sample.
It is no~ understood that the presence of cyanide in either the i.on exchange partlcle treatment sol~tion or the buffer solution will in-crease the incidence of a tirr.e-factored conversion of ferrous hemoglobin deri.va~ives to errihemo~
globin cyanide, with an attendant alteration in the integer or value obtained by spectromet-ric analysis.
Of course, a skilled laboratory ~erson o-r techni-cian would (and wiLl) compensate for such varia-tion. However, ~rovision o~ a standard test -that can (and wîll) be ~erfor~ed by relatively unskille(l personnel mandates the u.se of a techn:ique ancl pro-cedure which does not em210y cyan:ide.
Other advant..l~e~s o~ rlon-(lse o~ cyanide w:il.l incl.ude the Eacil:LtatLon of export-:irrlport:
of mi.crocol~l~ms and rea~,ents accorcling ta the :inven-tion. In many countries throu~hout the world~
labeling of a ~roduct as containin~ cyanide (even in trace amounts) presents reg1l1atory complications.
In countries of Droduct manu:Eacture, eliminati.on oE
wastes or residue re:Lating to either reagen~ pre-paration or ion exchange material equilibra~ic)n can (and will) give cause to environmental llaYclrd situat:ions and legit:imate gc~vernmental entity con-cern. Finally, disposal of used colurnns and reagent solutions is simpLif:ied.
DISCLOSURE OF INVENTION
It is an object of the invention to ~ro-vide methods to determine a nu~erical percentage value as a diagnostic indicator of the blood sugar , ~ .
. : . ,; :
.
condition of a speclfic person, witho~lt the use o.E
reagents or solutinns containing any amounts, trace or otherwise, of any cyanide compound ~r anion C~ .
It is a further object to provide a S method which will separate, detect and measure the ratio of the sub-group of hemog]obitl species Hb-A-la c to the total hemoglobins (Hb) present in the blood of a specific person quickly, inexpensively, accur-ately and without the presence of a cyanide com-pound or the anion CN .
It is still further an object to provide a method which, a].though reyuiring oE a number of sequential or consecutive steps, is of such a charac-ter and nature as to permit the adopt:ion o.E pr()-cedures arlcl pro~ocoLs ~Ih3.ch may becorne st.ln~ (l .. nn(l ro~ltlne, p~rm:itt:in~ per.son~ sk:i.lled in the a-r~ o.f.
c1inical. chem:lstry to rcpeat:ecll.y an~l cncc~lr~:lt:eLy test the bloocl o.E large groups of persons to estab-lish a data base for use by qualified, specialized and medically trained personnel in diagnosing the blood sugar condition of specific persons suspected as being diabetic.
It is still another object of the inven-tion to provide microcolumns, for chromatographic clinical techniques and procedures to determ:ine an lndlcation or level. of the hemo~rjl.o~:in species ~Ib-~la c having no cyanlcle therein but: h.lv:ing prede~-termineclrnicrochrornatograph:Lc char.lcter:istics and properties for practice of the rnethods according to the invention.
These and other objects of the invention, as well as the advantages thereof, will be apparent in view of the detailed descriptions of the various embodiments thereof as set :Eorth below~
In general, the methods according to ~he invention to determine a numerical percentage value as a diagnostic indicator of the blood s~gar condi-tion of a specific person use a whole blood sample taken from such person and -thereafter prepared as a test sample containing a red b3ood cell hemoly-sate solution. Thereafter, a quanti~y of the test sample is introduced into an end of a column bed having no cyanide therein which will absorb hemo-globin species present in sa;d test sample. The column bed comprises an equilibrated s~lspension of ion exchange material particles havlng a size less than 100 mesh, The particles in a particular column bed are one member selected from the class consisting of CarX~ arl(l CarYOIl, "Car" represents an inert substrat:e Eor carryi.~ .i.oni.~,al)Lcl gro~ll)s X
l.S providing c~ oc~ ed ca!:.i.ons ll arld iotl:iz~)l.)Le ~,,roup~ Y~ provi~ling di~ns.soci.at~(l r.lniot-~c; 01~ .
I~ ha9 been detertll~ined as cri~i.cr.ll.and essential for the practice of the invention that the CarXH particles be weakly acidic cation ex-changers and have a reported pKa: 3-7 and be used in an equilibrated suspension at a pH: 6.0-75 al:
22.5C.
It has also been determi.ned ~s criticrl:l and esse~ 'L ror ~ l~c~ l-r<lc~: i.cc ol~ tl~ velll:. i.OI~
tha~ the CarYO~l parti.cles be wealil.y hLI~;i.c ~Ini.on exch~mger.s cltlcl llave .~ report.ed pK,I: 7-1.0 r.lnd be usecl in an equi'l.ibrated suspens.ion at a p~l: 7.'3-9.0 at 22.5C, Therea:Eter a quantity of a buEfer solu-tion having no cyanide therein is introduced into an end of the column bed to preferentially elute therefrom a first fraction which contains certain of the hemoglobin species present in t~e test sample. t~hen the column bed is an ec~ui.Li.brc.Lted suspension of CarX~I particles, the ~irst :Frc.lct-ion " ~.$~
will contain substantially all of the hemoglobin species Hb-Ala c present in the test sample. ~hen the column bed is an equilibratecl suspension of CarYOH particles tne first fraction will contain essentially all of the ~hemoglobin species present in the test sample other than, to the exclusion of, Hb-A]a c In either event~ an al.iquo~ quantity o~
a first eluate fraction is collected :Erom the other end of -the column bed.
In the ~irst method embodiment of the in-vention, a quantity of a wash solution is lntro-duced into an end of the column bed ~o desorb and elute therefrom a second fraction ~hich will con-tain substantially all o:E the remaining hemoglobin lS species present in the test sampl.e. ~hen ttle column bed :is an equilibrated suspeTIsion oE CarXtl pc~rt:icles, the c;ecorl(l I:'ract:ion w:i. I.L c.~ont:cl:i.rl es~qcr~ y a L ~
~h~ hemo~:Lob:lrl species preserlt ln the tes t sanlp.l.e~
other than, to the excluslon o.E, Hb-Ala c ~Jhen the column bed is an equilibrated suspension of CarYOH particles, the second fraction will contain s~bstantially all of the hemoglobin species Hb-Ala c present in the test samp:le In ei~her event, an aliquot quantity of ~ second e:Luate fraction is collected from the other end of the column bed.
Then, tlle hemoglobin speci.es present in the .ELrst and second eluate fractions are separately detected and measurecl ~y s~ectrc~metric an~nlysi.s (color) and the resrJective ar.~ounts ~hereof are ex-pressed as numerical values ~h.ich are then compared in accordance with a mathematical Eorrnula.
In the second method embodiment of the invention, using a column bed having an equilibra-ted suspension of either CarXH or CarYOII particles and a buffer solution, an aliquot quanti~y of a first eluate frac~ion is collected from an end of the column bed. Also, a quantity of a test sample is significantly diluted to provide a red blood cell hemolysate solution ~hich m,~y be conveniently detected and measured by spectrome-tric analysis.
Then, the hemoglobin species present in the first eluate fraction and in the diluted hemolysate sol-u-tion are separately detected and meas~red by the spectrometric analysis (color) and the respective amounts thereoE are expressed as numerical values which are then compared in accordance with a rnathe-matical formula.
In e:ither method e~bodiment, iln appro-priate mathematical ~ormuLa wlll provi(le a nullleri-L5 cal percentclge vaLuc Eor thc hemogLobln sp~cies ~Ib-Ala c present in ~ particuLclr te,C;t sampLe Eor use as a diagnostic indicator of the blood sugar characteristics of the person providing that test sample.
In all embodiments of the inVentiOTl, the resultant numerical percentage value is available for use by qualified, specia:Lized and ~edically trained personnel as a diagnostic indicator of the blood sugar characterist:ics oE tile patien~ p-rovicl-ing thc test sample.
A microcolumn Eor use in the pract:ice of either embodiment o~ ~he inventiorl has a reservoir discharging into a barrel terminating in a dis-charge tip. The junctures between the reservoir and barrel and the barrel and discharge tip are closed by ~ransverse discs. Ion exchange parti-cles positioned in the barrel between ~he discs provide a column bed. The discs are permeable to a red blood cell hemolysate solution. The column bed comprises an equilibrated sus~ension of parti-.
.
cles having no cyanide therein. The particles will have a size of less than 100 mesh and in a particu-lar column will have one member selected from the class consisting of CarX~. and CarYOH.
BRIEF_DESCRIPTION OF ~RAI~TII~GS
The drawing schematically shows practice of the invention s~ecifically as to the first method embodiment wherein first and second fractions of hemoglobin species are eluted from an improved chromatographic microcolumn shown substantlally in full scale.
BEST MO ~ FOR C R YING OUT 'l'llt~'. tNV~l'lON
Practice oL~ tLIe me~hocl .:lccord:i.ng ~.o ~he lnventl.orl reclulres tlle coL:Lec~i.on o~ ~1 whol.e bloocl sample from a person or patienr.. The whole blood sample may be taken using conventionaL clinical chemistry techniques and procedures.
The disclosure o~ the Isolab-Acuf~ prior art patents describing in detail two general sl:a~e of the art procedures for preparing a suitab~Le test sample, referred to generally by the numeral 10 containing a red blood cell hemolysate solution Erom a whol.e bloocl samp:le, I.s :incorporatecl herc:iTl by reEerence. A proceclurc LO-a is used to prepclre a test sample LO which is predominantly the hemo-globin content of the whole blood sample. A pro-cedure 10-b is used to prepare a test sample 10 which may include ~he plasma proteins, lipids~ and -the white and red blood cell debris, in addition to the hemo~lobin content of the whole blood sarnple.
~lith reference to the drawing, which is substantia].ly in full scale, a chroma-tographic microcolumn is indicated general'Ly by the numeral 20. A column 20 comprises a reservoir 21 discharg-ing into a barrel 22 ter~inating in a discharge tip 23 selectively closed by a cap 24 The juncture or intersection between the rese]rvoir 21 and the barrel 22 is closed by a transverse plate or disc 25. The juncture between the barrel 22 and the dis-charge tip 23 is also closed by a transverse plate or disc 2~. The ion exchange materiaL particles comprising the column bed between the discs 25 and 26 are referred to generally by the numeral 27.
Each retaining disc 25 and 26 is permea-hle, h.lving a networl; o~ mi.c-ropores perm~ irlg introduc~lon oE a red b'lood ce'Ll hemoly~;ate so':Lll-tion ~rom res~rvo:ir ~l into the barreL 22, arld re-moval of an clu.lte :Eract:ion :~rom the barrc!.l. 22 through the tip 23, while retaining the colulTm bed of particles 27 within the barrel 2~. The discs 25 and 26 may be made from a conventional flexible, resilient, linear, high density polyethylene o~ the Ziegler type. Com~ercially, th:is type oE fllter grade polyethylene is produced and sold under the name of Vyon.
According to the inVentiQn, t'he ion ex-change materl.al parti.cles 2'7 have a si.æe less than 'l00 mesh and are selectcd ~rom the class consisting of Car~H and CarYO~I. "Car" represents an inert substrate and may be cellulosic or a resin copoly-mer oE polystyrene or rnethacrylic acid and divinyl-benzene. X represents an ionizable group provid-ing dissociated cations H~ carried by a "Car" and may be a carboxyl group or a carboxymethyl group.
Y represents an ionizable group providing disso-ciated anions OH carried by a "Car" and may be a .
~. .. .
diethylaminoethyl group or a mix~ure of amine groups having the general for~ulae - NH,, N~.R ancl M(R)2.
The CarXH particles 27 may also be charac-terized as weakly acidic cation exchangers having a reported pKa: 3-7. The CarYOH particles 27 may also be charac~erized as weakly basic anion ex-changers having a reported pKa:7-lO.
The commercially available form of ion exchange material particles 27 will usually require preparation or ~rea~.ment for use ln a barrel 22 of a microcolumn 20 between the discs 25 and 26. Such treatment could be perEormed with the particl.es 27 in situ in the column barrel 22. Ilowever, i.t i.s pre~errecl thnt the particL,es 27 ~.'or ~I, scri.e(; of `L5 .i.clenticaL columnc; 20 be t.reate(l us:ing a ~at:ch ~chnl.~lu~, wh:L~Il wi.Ll pe~rlrlLL t:he usc o~ .).la.lmrll: 2() havi.n~ preclel:ermine(l mi.crochromatog,r~ll)h:ic ctl~lrac-teristics ancl properties.
The ion exchange material particles 27 constituting the column bed of a column 20 are used in the form of an equilibrated suspension 27(S) h,aving a predetermined or "starting" pH. The CarXH
particles are used in a suspension 27(S~ at a pH:
6.0-7.5 at 22.5C. The CarYOll ~art-icl.c~s .Ire ~Ised in a suspension 27(S) a~. a p~-l: 7.'3-9.0 at 22.5C.
A suspenslon 27(S) :Ls prepared us:ing a treat:ment solution 2~, having no cyanide therein.
According ~o the invention, a treatment solution 28 may also be used as buffer solution 128 for elution from a column bed, comprising a sus-pension 27(S) of either CarXH or CarYOH particles, oE an eluate fraction containing certain, but not all, of the hemoglobin species present in the test sample 10.
A quan~i-ty of a test sample 10 prepared by either procedure 10-a or 10-b is introduced into one end of a column 20 having a coltlmn bed com-prising an equilibrated suspension 27(S). A test sample prepared according to procedure 10-a will require a 1:4 dilution using dislilled water.
Preferably, the column 20 is positioned vertically, the discharge tip cap 24 is removed and a predetermined volume of a test sample 10 is dis-charged or placed into the reservoir 21. A mclJor portion of the test sample 10 will pass readil.y through the disc 25 and onto t~le column bed ol the suspension 27(S). The minor portion of the test sample 10 remaining on or i.n the disc 25 slloul.d be purged or clisplaced onto the co:L~Irnn bed usinF~ a small volume (e.g., 0.2 ml) of a soLu~:i.on :in~ (k!cl :~or u~e ac; a buEfer ~oLut::ion 128 in tlle n~-~xt se~luell-tial metllocl ~;teE) accorclinf~ to t~e :invent:iorl.
A predetermine~ or aliquot quantity o.E a buffer solution 123 having no cyanide therein is dis-charged into the column reservoir 21 to preferential-ly elute from the discharge tip 23 a first .Eraction 30 of the test sample lØ The fi:rst fraction 30 i5 collected in a receiver 31. I~hen the column bed i.s a suspension 27(S) of CarX}I particles, the fi.rst fraction 30 ~ill contain substantially all of the hemoglobin specie~ llb-~La c present i.n ~.he test sample 10. When the colunm bed :is a suspension ~7(S) o Car~OH partic:les, the first fraction 30 wi:Ll con-tain essentially all of the hemoglobin species pre-sent in the test sample 10 other than, to the ex-clusion of, Hb-Ala c In either event, after a period of time following addition of the buffer 28, an aliquot quantity of a first fraction 30 will be collected in a receiver 31.
In the firs-t method embodiment of the in , : ~
.,, ' ' ~, $ 7 vention, as shown in the drawing, a second fraction 32 of the test sample 10 is collected. in a receiver 33 after an aliquot quanti~y of a wash solution 34 is discharged into the col-umn reservoir 21. The S second fraction 32 will contain subs~antially all of the remaining hemoglobin species present in the test sample 10. ~hen the column bed is a suspension 27(S) of CarXH par~icles, the second fraction 32 will contain essenti.ally all of the hemoglobin spe-cies present in the test sample 1.0 other than llb-Ala c ~Jhen the column becl is a suspension 27~S) of CarYOH par~icles, the second fraction 32 will con-tain substantially all of the hemoglobin species Hb-Al~ c present in the test sampl.e 10. In either event, an alicluo~ uant~ y o~ a. secorlcl Eract.ion 32 will be co~ cte~ i.n a r~ceI.ver 3~.
~r~,~. wash so:l.ut:.i.on ~4 w:ill. hllve llO ey~l~li.(le~
th~reln. Otherwise, ~ precise .~orn~ula oE a wasll solution 34 is not critical, so long as use thereof will not alter or modify the spectrometric absorp-tion characteristics ("color") of an elute fractlon 32. A compatible wash sol.ution 34 will have either ionic strength or relative pH suEficient to f~ Ly or completely desorb substantiaLly all the rem11n-ing blood components of a test sample 10 Erom a column bed of ion exchange material particles 27.
For example, 1 four milliliter (4 ml) vol~lme of l~M
NaCl m&y be discharged into a colurnn reservoir 21.
After a period of time (e.g., 20-30 minutes), an eluate fraction of substantiaLly 4 ml volume will be collected in a receiver 33. A fraction 32 eluted from a suspension 27(S) of CarXH particles will require of suitable dilution using distillecl water prior to the next sequential methocl step according to the invention.
According to both method embodiments of the invention, the determination of tlle ra-~io of Hb-Ala c to the to~al hemoglobins (Hb) present in the whole blood sample collec-ted from the person or patient uses spectrometric apparaLtus referred to generally by the numeral 40, following performance of liquid column microchromato~raphic techniques and procedures using a test sample 10.
The spectrometric analysis is perEormed by an apparatus 40 which measures absorption of li~ht caused by the hemoglobin species present -in the test sample lO. It is known ~hat the visible portion of the spectrllm for cletectin~J the preC;ence 0 f rl tle mo ~ l ob :irl L s :in ~ e vi.ole~ r an ~.~,c.~ 0 ~
LS speci~:lcal.l.y, at s~ st~n~::Lally ~5 tlm ol ~L5() ~.
Th~ aL~paratus ~t) may be an c)pt i..cr~ JI)ec~-trometer "~cclic-lte(l" or pre-set at the seLectecl wave length of 4l5 nm. The apparatus 40 may also be a spectrophotometer, a form of spectrometer with associated equipment which supplies the ratio, or a function of the ratio, of the radiant power of two beams as a function of an acljustably selec-ted spectral wave length. Because the spectrome-tric analysis accorcling to the invention :is ~or the purpose of detecting ancl measuli.n~ llen~o~.~:lobi.n species Erorn the test .sampLe 1.0 by li.~ht absorE~-tion characteristics, altcrnative Forms of appara-tus 40 could b~ used; for example, visual compara-tors such as a set of Nessler tubes.
In the first method embodiment of ~he in-vention, the contents of the receivers 31 and 33 are individually transferred into appropriate cu-vettes for the spectrometric apparatus 40. Spec-trometric analysis of the first and second eluate fractions 30 and 32 will provide integers or , natural nwmbers which will express, represent or indicate the amounts of hemoglob-in species present in the test sample 10. When using a conventional spectrometer or spectrophotomete:r as the apparatus 40, the displayed interger is a function of the absorbance ~A), a measurement of the amount of light of the spectral wave leng-th of 415 nm absorbed by the hemoglobin species during passage through the cuvette and toward the sensing photocell.
The expressed numerical -values for the separately detected and measured hemoglobin species in each eluate fraction 30 and 32 oE a par-ticular test sample 10 are then compared in accordance with a mathematiccll formula.
When the eLuate ;~ractic)ns 30 arld 32 are eluted ~rom a colu~m hecl comprisln~, a ~nlsl)en-sLon 27(S) o~ CarXH par~lcles, thc comp~a~ioLl Ls inte~er for firs-t fraction 30 x 100 =
integer for first fraction~30 -~
integer for second fraction 32 a numerical percentage value.
When the eluate Eractions 30 and 32 are eluted from a column bed comprising a swspen-sion 27(S) of CarYO~I particles, the c(mlputclt:io nt~ or ~:~cond_Lraction 32 x 100 integer i-or ~rirsl~ fraction 3() -~
integer Eor second fraction 32 a numerical percentage value.
In the second embodiment of the inven-tion, using a column bed comprising a suspension 27(S) of either CarXH or CarYOH particles and a buffer solution 128, analiquot quan~ity of a first eluate fraction 30 is collected in a receiver 31.
Also, either at a prior time, concurrently or con--17~
secutively, a quantity of the test sample 10 is prepared as a red blood cell hemolysate solution 42 which may be conveniently det,ected and measured by spectrometric analysis. It will be apparent that the light absorption characteristics of -the test sample 10, without significant dilution~ would be of such magnitude as to impair operation e~ficiency of the sensing photocell of conventional spectro-metric apparatus 40. Accordingly, and by way of example, a quantity of test sample 10 equal to the volume of test sample 10 introduced into an end of a column 20, prior to elution oE the first ~raction 30, showld be cliluted using distilled water in the ratio substantially l:l~8o to prepare a hemolysate lS ~solution 42 Eor analysl~ by a ~:pcctrome~ric aE~para-tus l~O.
The exl)resse.d n~lmerica'l. Vcll.Ue~ Eor the separately cletecte~ atld measured hernoglobLn spec:ies in the eluate fraction 30 and in the diluted herno-lysate solution 42 oE a particular test sample lO
are then compared in accordance with a mathemati-cal formula.
When the eluate fraction 30 is eluted from a column bed comprising a suspells:Lon 27 (S) of CarXH particles, the computation is integer for eluate ~raction. 30 x 100 in te ger for flelTIo'r--ate ~sol~
a n~erical percentage value.
When the eluate fraction 30 is eluted from a column bed comprising 27(S) of CarYOH par-ticles, the computation is (inte~er for eluate fraction 30 x 100) _ ~ ~ (integer for hemolys'ate'sorution ~
a numerical percentage value.
Use any of these mathematical compu--tations will provide a nwmerical value :Eor the hemoglobin species Hb-Ala c present in a particu-lar test sample 10 for use as a diagnostic indica-tor of the blood sugar character:istics of -,he per-son providing that test sample.
The -following Examples will ~urther illustrate and describe the practice of t~e inven-tion by persons skilled in the art of clinica]
chemistry.
Exarnple 1 A CarYOH ion exchange material particle 27 may be of the type wherein "Car"is cellulosic and Y~ Ls a die~hylaminoethy:L group, -O-Cll2-C~l2-N(C~l5~2, having a reported pK,I oE substanti~ll.Ly 9.5. Commerckllly, ~his lype oE iOLI (~xch~ e m~
terial ls sol(l under the ~rlme Whatman DEAE-52.
These cellulose particles 27 are pre-pared as a suspension 27ts) having a pH of sub-stantially 8.5 at 22.5C by mixing with a "tris"
treatment solution 28 of -the fo~lula 6.06g H2N.C
(CH2OH)3 (0.05 M), with 0.10g NaN3 (0.01%) as a preservative, made up in one liter of H20. After mixing with the treatment solution 28, the cel:lu-lose particles 27 are then Eurther treated w:ith an acid solution 29 (e.g., 4 M HCl) to readjust to the pH of substantially 8.5.
In both method ernbodiments of the in-vention, a quantity of a buffer solution 128 is introduced into an end of a column bed of a sus-pension 27(S)of these cellulose particles to pre-f~rentially elute therefrom an eluate fraction 30 containing essentially of the hemoglobin species present in a test sample 10 other than Hb-Ala_C.
The buffer solution 128 may be a "tris" solution of the same formula as the -treatment solution 28 wlth the pH adjusted to substantially 7.7 at 22.5C
using concentrated HCl.
' ,, ~
Another CarYOH ion exchange material particle 27 may be of the type w'herein "Car" ls a resin copolymer of polystyrene and divinylbenzene and Y+ is a mixture of primary, secondary, and ter-tiary amine groups having the general :Eormulae -NH2, N~'~ and N(R)2, where R is an aliphatic hydro-carbon radical such as -CH3 or -C2H5. These par-: ticles 27 have a reported pKa: 7-9. Cor~mercially, this type of ion exchange material is sold under the names Dowdex M~A-l and Amberlite IRA-93.
These resi.n part:ic'les 27 may be pre-pared as a suspension 27(S) llaving a pl~ OL s~lb-stanti~'lly 7.5 a~ ~.2..5C ~y m:l.xitlg wi.~ c~ ri.~
trcatment ~o'lu~Lon 28 o.E the ~ame .f.'orm-lLcl flS UXCIII1-ple 1.
A first fraction 30 of a .est sample 10, containing essentially all of the hemoglobin species other than Hb-Al,l c' may be elu~ed from a column bed of a suspension 27(S) o~ these resin particles by use of a "~ri.s" bufEer so.lLl~:ioll 1.28 of the same formula as in ~xample 'l, w.i~h a pH ad-justed to substantially 7.5 at 22.5C.
Exame~e 3 A CarXH ;.on exchange material parti-cle 27 may be of the type wherein "Car" i5 a resin copolymer of methacrylic acid and divinylbenzene and X+ is a carboxyl group, -COOH, having a reported pKa: ~-6. Commercially, this type of ion ex-change material is sold under the name Amberlite , :. ' " ' ' ~ ' ' :, ', ' : . , . .. - : .
:.: ' : ' .. , ' ' .' . ', ,, . ', ., :
The resin polymers 27 are prepared as a suspension 27(S) having a pH of substantialy 6.98 at 22.5C by mixing wi~h "phosphate" treatment solution 28 of the formula: 3.74g KH2PO4 (0.027M) 7 0.955g KOH (0.017 M), with 0.10g NaN3 (0.01%) as a preservative, made up in one liter of H20. After mixing with the treatment solution 28, the resin particles 727 are then further treated with an acid solution 29 (e.g., 4 M H3PO4) to readjust to the pH of substantially 6.98.
In both method embodiments of the inven-tion, a quantity of a bu~fer solution 128 is intro-duced into an end of a column bed of a suspenslon 27(S) of these resin particles to preferentially elute therefrom an eluate fractlon 30 containing substantially aLl of the hemoglobi-n species Hb-Ala c present ln a test sample 10. The buffer so lutic)n 128 may be a "phosphate" solutioll o~ th~
same orm-l1a as the treatment solutlon 28 antl hav~
ing a pH of substantially 6.98.
Examp~e 4 Another CarXH ion exchange material particle 27 may be of the type wherein "Car" is cellulosic and X is a carboxymethyl group, -O-CH2-COOH, having a reported PKa of 3.5. Commer-clally, this type of ion exchange mater:ial :i.s solcl under the nam~ Whatman CM-52.
These cellulose particles 27 may be prepared as a suspension 27(S) having a pH of sub-stantially 6.8 a~ 22.5C by mixing with "phosphate"
treatment solution 28 o~ the formula: 3.74g KH2PO4 (0.027 M), and 0.748g KO~ (0.013 M), with 0.10g NaN3 (0.01%) as a preservative, made up ln one liter of H20.
,. . - . :
, $ ~
A first frac-~ion 30 of a test sample 10, containing substantially all of ~he hemoglobin species Hb-Ala_C, may be eluted from a colu~n bed of a suspension 27(S) of these cellulose particles by use of a "phosphate" buffer solution 128 of the same formula as -the treatment solution 28 and having a pH of substantially ~.8.
_Example 5 The cellulose particles 27 o:E Example 4 may be prepared as a suspension 27(S) having a pH of substantially 6.1 at 22.5C by mixing with a "bis-tris" treatment solution 28 of the formula:
6-28g ~HOCH2c~l2)2 NC (Ctl2O~1)3 ~0.03M), with O.lOg lS NaN3 (0.01%) ~s a preservatlve, mad~ up i.n one liter o~ ~l20. ~Eter mixLng, wi~h the treat:merlL- ~o:lalti(3n 28, the cellulose particles 27 are ~hen fuL~:her treated with an acid solution 29 (e.g., 4 M HC]) to readjust to the pH of substantially 6.1.
A first fraction 30 of a test sample 10, containing .substantially all of the hemoglobin species ~Ib-AIa c~ may be eluted from a column bed of a suspension 27(S) of these cellulose part:icles by the use of a "bis-tris" buffer solution l28 of ~5 the same formuLa as the treatment solutlon 28 with the addltion of 2.34g NaCl (0.04 M) ~ncl having a p~l of substantially 6.1.
Example 6 The resin particles of Example 3 may be prepared as a suspenslon 27(S) having a pH of substantially 6.8 at 22.5C by mixing with a "bis-tris" treatment solution 28, of the same formula as in Example 5. A first fraction 30 of a tes-~
sample 10, containing substan-tially all of the hemoglobin species Hb-Ala c~ may be eluted from . . ..
, ' ~,'; ,' ' . ' ' '"
': ' .. ,, ' ' a column bed of a suspension 27('~) of these resin particles by the use of a "bis-trisll buffer solu-tion 128 of the same formula as t:he treatment solu-tion 28 with the addi~ion of 7.02g NaCl (0.12M), having a pH of substantially 6.8.
SU~ARY
In all embodiments of the inven-tion as described, several steps, techniques or proce-dures are disclosed wherein dilution, usin~ dis-tilled water, is either required or suggestecl. It will be undexstood by a person skilled in the art of clinical chemistry tha~ the best modes of prac-ticing the invention usin~, an i.rnproved IllicrocoLurnn 20 will recluire careElll a(loptl()rl an~l corlsis~:c?nL
followlng of rout:ine proce(lures, i.E ~lle inVerl~
i~s tt) xepre8ent a rel kLbl~e metho(l of assess.in~
the presence oE d:Labetes and monitoring the degree of diabetic control. It will be further understood by a practitioner of the invention that a procedure or protocol for repetitive testing of large numbers oE persons, both diabetic and normal, will inherent~
ly incorporate therein: standard quantities and volumes of test samples 10, solutions 28 and ]28, 29 cmd 34, Erac~ions 30 and 32 and sol-l~ion 42;
conslstent and compatible dilut:ion ratios; and, care~ul select:ion and re~ lation of the sl~ectro-metric apparatus 40. There~ore, the Eull scope and extent of the invention should be determined solely by the words of the claims appended hereto.
.. . ~ .
' ' '
Claims (15)
1. A method to determine a numerical percentage value as a diagnostic indicator of the blood sugar condition of a specific person, wherein a whole blood sample is taken from said person and thereafter prepared as a test sample containing a red blood cell hemolysate solution, and there-after, a quantity of said test sample is intro-duced into an end of a column bed having no cyanide therein which will absorb hemoglobin species pre-sent in said test sample, said column bed compris-ing an equilibrated suspension of ion exchange ma-terial particles having a size less than 100 mesh, said particles in a column bed being one member selected from the class consisting of CarXH and CarYOH, where "Car" represents an inert substrate for carrying ionizable groups X- providing disso-ciated cations H+ and ionizable groups Y+ provid-ing dissociated anions OH- , said CarXH particles being a weakly acidic cation exchanger having a reported pka: 3-7 and being used in an equlibra-ted suspension at a PH: 6.0-7.5 at 22.5°C, said CarYOH particles being a weakly basic anion ex-changer having a reported pKa: 7-10 and being used in an equilibrated suspension at a PH: 7.3-9.0 at 22.5°C, and thereafter, a quantity of a buffer solution having no cyanide therein is introduced into an end of said column bed to preferentially elute therefrom a first fraction which contains certain of the hemoglobin species present in said test sample, a column bed which is an equilibrated suspension of CarXH particles, providing a said first frac-tion containing substantially all of the hemo-globin species Hb-Ala-c present in said test sam-ple, a column bed which is an equilibrated suspension of CarYOH particles providing a said first fraction containing essentially all of the hemoglobin spe-cies present in said test sample other than Hb-Ala-c, and thereafter, an aliquot quantity of said first eluate fraction is collected from the other end of said column bed, and then, A quantity of a wash solution is intro-duced into an end of said column bed to desorb and elute therefrom a second fraction containing sub-stantially all of the remaining hemoglobin species present in said test sample, a column bed which is an equilibrated suspension of CarXH particles providing a said second fraction containing essen-tially all of the hemoglobin species present in said test sample other than Hb-Ala-c' a column bed which is an equilibrated suspension of CarYOH parti-cles providing a second fraction containing sub-stantially all of the hemoglobin species Hb-Ala-c present in said test sample, and then, an aliquot quantity of said second elu-ate fraction is collected from the other end of said column bed, and then, the hemoglobin species present in the said first and second elute fractions are separate-ly detected and measured by spectrometric analysis and the respective amounts thereof are expressed as numerical values which are then compared in accordance with a mathematical formula to provide a numerical percentage value for the hemoglobin species Hb-Ala-c in said test sample for use as a diagnostic indicator of the blood sugar charac-teristics of said specific person.
2. A method to determine a numerical percentage value as a diagnostic indicator of the blood sugar condition of a specific person, where-in a whole blood sample is taken from said person and thereafter prepared as a test sample contain-ing a red blood cell hemolysate solution, and thereafter, a quantity of said test sample is intro-duced into ah end of a column bed having no cyanide therein which will adsorb hemoglobin species pre-sent in said test sample, said column bed compris-ing an equilibrated suspension of ion exchange material particles having a size less than 100 mesh, said particles in a column bed being one member selected from the class consisting of CarXH and CarYOH where "Car" represents an inert substrate for carrying ionizable groups X- providing disso-ciated cations H+ and ionizable groups Y+ providing dissociated anions OH- , said CarXH particles being a weakly acidic cation exchanger having a reported pKa: 3-7 and being used in an equilibrated suspen-sion at a pH: 6.0-7.5 at 22.5°C, said CarYOH parti-cles being a weakly basic anion exchanger having a reported pKa: 7-10 and being used in an equili-brated suspension at a pH: 7.3-9.0 at 22.5°C, and thereafter, a quantity of a buffer solution having no cyanide therein is introduced into an end of said column bed to preferentially elute therefrom a first fraction which contains certain of the hemo-globin species present in said test sample, a column bed which is an equilibrated suspension of CarXH particles providing a said first fraction containing substantially all of the hemoglobin species Hb-Ala-c present in said test sample, a column bed which is an equilibrated suspension of CarYOH particles providing a said first fraction containing essentially all of the hemoglobin spe-cies present in said test sample other than Hb-Ala-c and thereafter, an aliquot quantity of said first eluate fraction is collected from the other end of said column bed, and then, a quantity of said test solution is sig-nificantly diluted to provide a red blood cell hemolysate solution which may be conveniently de-tected by spectrometric analysis, and then, the hemoglobin species present in said first eluate fraction and in the said diluted hemolysate solution are separately detected and measured by spectrometric analysis and the respec-tive amounts thereof are expressed as numerical values which are then compared in accordance with a mathematical formula to provide a numerical per-centage value for the hemoglobin species Hb-Ala-c in said test sample for use as a diagnostic indica-tor of the blood sugar characteristics of said spe-cific person.
3. A column bed for use in the method of either claim 1 or 2 comprising a suspension of CarYOH
particles at an equilibrated pH of substantially 8.5 at 22.5°C and wherein "Car" is cellulosic and Y+ is a diethylaminoethy group.
particles at an equilibrated pH of substantially 8.5 at 22.5°C and wherein "Car" is cellulosic and Y+ is a diethylaminoethy group.
4. A column bed for use in the method of either claim 1 or 2 comprising a suspension of CarYOH particles at an equilibrated pH of sub-stantially 7.5 at 22.5°C and wherein "Car" is a resin copolymer of polystyrene and divinylbenzene and Y+ is a mixture of primary, secondary, and ter-tiary amine groups having the general formulae -NH2, NHR, and N(R)2, where R is an aliphatic hydro-carbon radical such as -CH3 or -C2H5.
5. A column bed for use in the method of either claim 1 or claim 2 comprising a suspension of CarXH particles at an equilibrated pH of sub-stantially 6.98 at 22.5°C and wherein "Car" is a resin copolymer of methacrylic acid and divinyl-benzene and X- is a carboxyl group.
6. A colum bed for use in the method of either claim 1 or claim 2 comprising a suspension of CarXH particles at an equilibrated pH of sub-stantially 6.8 at 22.5°C and wherein "Car" is cellu-losic ancl X is a carboxymethyl group.
7. A column bed for use in the method of either claim 1 or claim 2 comprising a suspension of CarXH particles at an equilibrated pH of substan-tially 6.1 at 22.5°C and wherein "Car" is cellu-losic and X- is a carboxymethyl group.
8. A column bed for use in the method of either claim l or claim 2 comprising a suspension of CarXH particles at an equilibrated pH of sub-stantially 6.8 at 22.5°C and wherein "Car" is a resin copolymer of methacrylic acid and divinyl-benzene and X- is a carboxyl group.
9. A microcolumn having no cyanide therein for use in the determination of a numerical percentage value as a diagnostic indicator of the blood sugar condition of a person providing a whole blood sample: having a reservoir discharging into a barrel terminating in a discharge tip, the junc-ture between said reservoir and said barrel and the juncture between said barrel and said tip each being closed by a transverse disc, said discs being permeable to a red blood cell hemolysate solution test sample prepared from said whole blood sample, and a column bed comprising an equilibrated sus-pension of ion exchange material particles having a size less than 100 mesh positioned in said barrel between said discs, said particles in a column bed being one member selected from the class consisting of CarXH and CarYOH, where "Car" represents an inert substrate for carrying ionizable groups X- providing dissociated cations H+ and ionixable groups Y+ pro-viding dissociated anions OH-, said CarXH particles being a weakly acidic cation exchanger having a reported pKa: 3-7 and being used in an equilibrated suspension at a pH: 6.0-7.5 at 22.5°C, said CarYOH
particles being a weakly basic anion exchanger having a reported pKa: 7-10 and being used in an equilibrated suspension at a pH: 7.3-9.0 at 22.5°C.
particles being a weakly basic anion exchanger having a reported pKa: 7-10 and being used in an equilibrated suspension at a pH: 7.3-9.0 at 22.5°C.
10. A column bed for the microcolumn of claim 9 comprising a suspension of CarYOH particles at an equilibrated pH of substantially 8.5 at 22.5°C and wherein "Car" is cellulosic and Y+ is a diethylaminoethy group.
11. A column bed for the microcolumn of claim 9 comprising a suspension of CarYOH parti-cles at an equilibrated pH of substantially 7.5 at 22.5°C and wherein "Car" is a resin copolymer of polystyrene and divinylbenzene and Y+ is a mixture of primary, secondary and tertiary amine groups having the general formulae -NH2, NHR, and N(R)2, where R is an aliphatic hydrocarbon radical such as -CH3 or -C2H5.
12. A column bed for the microcolumn of claim 9 comprising a suspension of CarXH particles at an equilibrated pH of substantially 6.98 at 22.5°C and wherein "Car" is a resin copolymer of methacrylic acid and divinylbenzene and X- is a carboxyl group.
13. A column bed for the microcolumn of claim 9 comprising a suspension of CarXH particles at an equilibrated pH of substantially 6.8 at 22.5°C and wherein "Car" is cellulosic and X- is n carboxymethyl group.
14. A column bed for the microcolumn of claim 9 comprising a suspension of CarXH particles at an equilibrated pH of substantially 6.1 at 22.5°C and wherein "Car" is cellulosic and X- is a carboxymethyl group.
15. A column bed for the microcolumn of claim 9 comprising a suspension of CarXH particles at an equilibrated pH of substantially 6.8 at 22.5°C
and wherein "Car" is a resin copolymer of metha-crylic acid and divinylbenzene and X- is a cabrboxyl group.
and wherein "Car" is a resin copolymer of metha-crylic acid and divinylbenzene and X- is a cabrboxyl group.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/090,319 US4238196A (en) | 1979-11-01 | 1979-11-01 | Methods to determine a diagnostic indicator of blood sugar conditions, and, liquid chromatographic columns therefor (cyanide free) |
| US090,319 | 1987-08-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1110467A true CA1110467A (en) | 1981-10-13 |
Family
ID=22222276
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA363,487A Expired CA1110467A (en) | 1979-11-01 | 1980-10-29 | Methods to determine a diagnostic indicator of blood sugar conditions and liquid chromotographic columns therefor (cyanide free) |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US4238196A (en) |
| JP (1) | JPS6236181B2 (en) |
| CA (1) | CA1110467A (en) |
| CH (1) | CH659527A5 (en) |
| GB (1) | GB2074466B (en) |
| WO (1) | WO1981001245A1 (en) |
Families Citing this family (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1168705A (en) * | 1980-05-27 | 1984-06-05 | Hamish Small | Ion exchange chromatography with indirect photometric detection |
| US4649122A (en) * | 1980-09-02 | 1987-03-10 | Leeco Diagnostics, Inc. | Means for determining percentage of glycohemoglobin in whole blood |
| US4407961A (en) * | 1981-03-16 | 1983-10-04 | Sanders James L | Ion-exchange system and method for isolation and determination of glycosylated hemoglobin in human blood |
| JPS58760A (en) * | 1981-06-25 | 1983-01-05 | Sekisui Chem Co Ltd | Separation of catabolic hemoglobin |
| US4468330A (en) * | 1981-04-27 | 1984-08-28 | Sekisui Kagaku Kogyo Kabushiki Kaisha | Filler for liquid chromatography useful for separating a hemoglobin variant in blood |
| EP0104165A1 (en) * | 1982-03-25 | 1984-04-04 | SANDERS, James Lynn | Ion-exchange system and method for isolation and determination of glycosylated hemoglobin in human blood |
| US4409335A (en) * | 1982-05-28 | 1983-10-11 | Bio-Rad Laboratories, Inc. | Method for eliminating glucose dependent Schiff base effect from hemoglobin A1 assay |
| US4448888A (en) * | 1982-08-24 | 1984-05-15 | Bio-Rad Laboratories, Inc. | Compositions and method for correcting temperature dependence of hemoglobin assays |
| US4935346A (en) | 1986-08-13 | 1990-06-19 | Lifescan, Inc. | Minimum procedure system for the determination of analytes |
| US4787971A (en) * | 1987-01-23 | 1988-11-29 | Alan Donald | Miniaturized column chromatography separation apparatus and method of assaying biomolecules employing the same |
| US4853338A (en) * | 1987-05-20 | 1989-08-01 | Technicon Instruments Corporation | Cyanide-free hemoglobin reagent |
| GB2220369B (en) * | 1988-06-10 | 1993-01-27 | Inst Of Child Health | Method for testing body fluids by low pressure liquid chromatography |
| AU624935B2 (en) * | 1989-04-10 | 1992-06-25 | Nippon Kayaku Kabushiki Kaisha | Method for quantitatively measuring sugar-alcohol, column and kit therefor |
| US5468380A (en) * | 1989-04-26 | 1995-11-21 | Nippon Kayaku Kabushiki Kaisha | Method for quantitatively measuring sugar-alcohol, column and kit therefor |
| US5807747A (en) * | 1989-06-13 | 1998-09-15 | Clinical Innovations Limited | Method and apparatus for determination of glycosylated protein |
| JPH0486554A (en) * | 1990-07-30 | 1992-03-19 | Tosoh Corp | Analysis method for sugar albumin |
| JP3515782B2 (en) * | 1992-03-04 | 2004-04-05 | アボット・ラボラトリーズ | Measurement of glycohemoglobin by fluorescence quenching |
| FI112545B (en) * | 1997-05-30 | 2003-12-15 | Nokia Corp | Method and system for predicting the level of a glycosylated hemoglobin component in a patient's blood |
| US6177009B1 (en) * | 1998-04-03 | 2001-01-23 | Macherey, Nagel Gmbh & Co. | Apparatus for treating biomolecules |
| US6458326B1 (en) | 1999-11-24 | 2002-10-01 | Home Diagnostics, Inc. | Protective test strip platform |
| US6525330B2 (en) | 2001-02-28 | 2003-02-25 | Home Diagnostics, Inc. | Method of strip insertion detection |
| US6562625B2 (en) | 2001-02-28 | 2003-05-13 | Home Diagnostics, Inc. | Distinguishing test types through spectral analysis |
| US6541266B2 (en) | 2001-02-28 | 2003-04-01 | Home Diagnostics, Inc. | Method for determining concentration of an analyte in a test strip |
| US6761855B1 (en) * | 2001-11-05 | 2004-07-13 | Biosearch Technologies, Inc. | Column for solid phase processing |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4142856A (en) * | 1977-12-02 | 1979-03-06 | Isolab, Incorporated | Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor |
| US4142857A (en) * | 1977-12-02 | 1979-03-06 | Isolab, Incorporated | Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor |
| US4142858A (en) * | 1977-12-02 | 1979-03-06 | Isolab, Incorporated | Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor |
| JPS5499496A (en) * | 1977-12-02 | 1979-08-06 | Isolab Inc | Method of deciding diagnosis index of blood sugar condition and its device |
| US4142855A (en) * | 1977-12-02 | 1979-03-06 | Isolab, Incorporated | Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor |
-
1979
- 1979-11-01 US US06/090,319 patent/US4238196A/en not_active Expired - Lifetime
-
1980
- 1980-10-22 CH CH4356/81A patent/CH659527A5/en not_active IP Right Cessation
- 1980-10-22 WO PCT/US1980/001411 patent/WO1981001245A1/en active Application Filing
- 1980-10-22 GB GB8108196A patent/GB2074466B/en not_active Expired
- 1980-10-29 CA CA363,487A patent/CA1110467A/en not_active Expired
-
1981
- 1981-12-02 JP JP81500132A patent/JPS6236181B2/ja not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| JPS57501937A (en) | 1982-10-28 |
| JPS6236181B2 (en) | 1987-08-05 |
| GB2074466B (en) | 1983-08-03 |
| WO1981001245A1 (en) | 1981-05-14 |
| CH659527A5 (en) | 1987-01-30 |
| US4238196A (en) | 1980-12-09 |
| GB2074466A (en) | 1981-11-04 |
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